Study the Mechanism of Nef Induced Endosome Re-Distribution and the Role in HIV Immune Evasion

Nef, a determinant of human immunodeficiency virus (HIV) pathogenicity, is required for the maximal virus replication and disease progression in vivo. The HIV persistency is attributed to the ability of Nef in immune evasion, including MHC-I downregulation, anti-apoptosis activity, FasL upregulation, and prevention of the formation of immune synapse. Nef also directly or indirectly modulates the expression of the level of many cell surface proteins to influence viral pathogenesis. So far, more than 20 cell surface proteins are up- or down-modulated by Nef, but how Nef can modulate these proteins at the same time is still obscure. My study indicates that the endosome in the Nef-expressing cell is concentrated at the perinuclear region rather than dispersed punctate distribution in control cells. Changing the distribution of endosome may result in global changes of vesicular trafficking pathway and modulating surface expression of proteins. Recent prelimentary data demonstrates NefEEEE65 and PxxP75 are involved in the Nef-induced endosome aggregation. Expression of p150glued shRNA disrupt the Nef-induced endosome aggregation, also reduced the efficiency to downregulate MHC-I. However, Zap70 activation by Nef is similar in either aggregated or dispersed endosome distribution. These results suggest that endosome distribution play a role in modulation the expression of many surface proteins by Nef, but may not be important in the activation of signaling pathway. In this study, the kinetics of vesicular trafficking related to aggregated endosome will be study in details, including the efficiency of protein transport to plasma membrane, the rate of endocytosis and recycling, and the rate of protein degradation. To understand the molecular mechanism, the cellular proteins interact with EEEE65 and PxxP75 motif and signaling pathway induced by these two motifs involved in Nef-induced endosome aggregation will be further study. The interaction between Nef and microtubule motor as well as Alix in the regulation of endosome aggregation will be also examined. Lastly, we will determine the role of endosome aggregation in the formation of immunological synapse and test the hypothesis that Nef induced endosome aggregation is a spatial regulation for TCR signaling pathway. These results will provide a better understanding of the Nef functions and pathogenesis of AIDS, at the same time it also provide a good model system to study protein trafficking and spatially regulation for signaling pathway.

Project ID:PC9902-1672
External Project ID:NSC98-2320-B182-026-MY2